Preeti Chhabra

Etiology of Viral Gastroenteritis in Children <5 Years of Age in the United States, 2008-2009

BACKGROUND Although rotavirus and norovirus cause nearly 40% of severe endemic acute gastroenteritis (AGE) in children <5 years of age in the United States, there are limited data on the etiologic role of other enteric viruses in this age group. METHODS We conducted active population-based surveillance in children presenting with AGE to hospitals, emergency departments, and primary care clinics in 3 US counties. Stool specimens from these children and from age-matched healthy controls collected between October 2008 and September 2009 were tested for enteric adenovirus, astrovirus, sapovirus, parechovirus, bocavirus, and aichivirus. Typing was performed by sequencing and phylogenetic analysis. RESULTS Adenovirus, astrovirus, sapovirus, parechovirus, bocavirus, and aichivirus were detected in the stool specimens of 11.8%, 4.9%, 5.4%, 4.8%, 1.4%, and 0.2% of patients with AGE and 1.8%, 3.0%, 4.2%, 4.4%, 2.4%, and 0% of healthy controls, respectively. Adenovirus (type 41), astrovirus (types 1, 2, 3, 4, and 8), sapovirus (genogroups I and II), parechovirus (types 1, 3, 4, and 5), and bocavirus (types 1, 2, and 3) were found cocirculating. CONCLUSIONS Adenovirus, astrovirus, and sapovirus infections were detected in 22.1% of the specimens from children <5 years of age who had medical visits for AGE and tested negative for rotavirus and norovirus. No causal role for parechovirus and bocavirus was found.

Epidemiological, clinical, and molecular features of norovirus infections in western India.

The study was conducted to investigate the molecular epidemiology of noroviruses (NoVs) from western India. A total of 830 fecal specimens were collected during July 2005–June 2007 from children, ≤7 years of age suffering from acute gastroenteritis in Pune, Nagpur, and Aurangabad cities. All the specimens were subjected to RT‐PCR, sequencing and phylogenetic analysis for detection and characterization of Genogroup I (GI) and GII NoVs. NoV positivity varied between 6.3% and 12.6% in different cities with the predominance of GII (96.6%). NoV infections were very common in the patients ≤2 years of age. A majority (55%) of the patients suffered from severe disease, however, vomiting was not experienced in 35%. Coinfections with rotaviruses were found in 10% cases. Summer month seasonality supported NoV infections in western India. The phylogenetic analysis of partial RNA polymerase and VP1 (capsid) genes identified 2 GI (GI. 2 and GI.6) and 5 GII (GII.4, GII.6, GII.7, GII.8, and GII.14) genetic clusters with possible occurrence of “2007 new‐variant” of GII.4. Six different combinations of RdRp and capsid genes (GII.b/GII.3, GII.b/GII.4, GII.d/GII.3, GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13) were also identified. GII.4 (52%) prevailed in 2005–2006 while the predominance of probable recombinant NoV strains (58%) was noted in 2006–2007 with the contribution of GII.b/GII.3 at 79% level. GII.b/GII.18 type identified in 37% infections in 2005–2006 was completely replaced by GII.b/GII.3 type in 2006–2007. This is the first report that highlights the norovirus epidemiology and strain diversity demonstrating possible circulation of new variants in patients with acute gastroenteritis from western India. J. Med. Virol. 81:922–932, 2009.

Diversity of circulating rotavirus strains in children hospitalized with diarrhea in India, 2005-2009.

BACKGROUND India accounts for 22% of the 453,000 global rotavirus deaths among children <5 years annually. The Indian Rotavirus Strain Surveillance Network provides clinicians and public health partners with valuable rotavirus disease surveillance data. Our analysis offers policy-makers an update on rotavirus disease burden with emphasis on regional shifts in rotavirus strain epidemiology in India. METHODS Children <5 years requiring hospitalization for acute gastroenteritis were selected from 10 representative hospitals in 7 cities throughout India between November 2005 through June 2009. We used a modified World Health Organization protocol for rotavirus surveillance; stool specimens were collected and tested for rotavirus using enzyme immunoassay and reverse-transcription polymerase chain reaction. RESULTS A total of 7285 stool specimens collected were tested for rotavirus, among which 2899 (40%) were positive for rotavirus. Among the 2899 rotavirus detections, a G-type could not be determined for 662 (23%) and more than one G type was detected in 240 (8%). Of 1997 (69%) patients with only one G-type, the common types were G1 (25%), G2 (21%), G9 (13%), and G12 (10%). The proportion of rotavirus infections attributed to G12 infections rose from 8% to 39% in the Northern region and from 8% to 24% in the Western region. CONCLUSIONS This study highlights the large, ongoing burden of rotavirus disease in India, as well as interesting regional shifts in rotavirus strain epidemiology, including an increasing detection of G12 rotavirus strains in some regions. While broad heterotypic protection from rotavirus vaccination is expected based on pre- and post-licensure data from other settings, effectiveness assessments and rotavirus strain monitoring after vaccine introduction will be important.

Molecular characterization of three novel intergenotype norovirus GII recombinant strains from western India.

The phenomenon of recombination has been widely described among noroviruses (NoVs) in the past few years. In a NoV surveillance study conducted in western India, 3 novel and 3 known combinations of RNA-dependent RNA polymerase (RdRp) and capsid genes were identified in genogroup (G) II NoV strains. The present study pertains to the characterization of three novel intergenotype NoV GII recombinant strains. RT-PCRs were carried out for the amplification of nearly complete RdRp and complete capsid genes spanning ORF1/2 overlap of three strains followed by sequencing of the amplicons. The recombination event was confirmed by phylogenetic analysis using Bayesian MCMC approach, SimPlot analysis and Maximum chi(2) method. Three novel intergenotype (GII) recombinations of GII.b/GII.18, GII.1/GII.12 and GII.3/GII.13 specificities were identified respectively in the strains PC03, PC24 and PC25 for the first time. The breakpoint in the novel recombinants was placed in the vicinity of the 20 bp ORF1/2 overlap, a common hotspot known to exist in NoV recombinants. The capsid genes of all of the 3 recombinants were closely related to their counter parts in reference strains however, a high degree of variation emerged in the polymerase genes especially of PC24 and PC25 in comparison to the reference strains.

Molecular characterization of a rare G1P(19) rotavirus strain from India: evidence of reassortment between human and porcine rotavirus strains

This study pertains to the characterization of a human rotavirus strain (NIV929893) with a rare specificity of G1P[19]. Three structural genes (VP4, VP6 and VP7) and one non-structural gene (NSP4) of strain NIV929893 were subjected to RT-PCR for amplification of entire coding regions. All of the amplicons were sequenced to carry out phylogenetic analysis. The complete amino acid sequences of the VP7 and VP4 gene products showed clustering of the VP7 gene with G1 strains of human origin and the VP4 gene with P[19] strains of porcine origin. The two viral proteins VP6 and NSP4, described previously as genetically linked proteins, were shown to be subgroup II and genotype B of human and porcine origins, respectively. The findings of this study provide evidence of reassortment between VP7/VP6 genes of humans and VP4/NSP4 genes of porcine species and an independent segregation of VP6 and NSP4 genes in a group A human rotavirus strain with G1P[19] specificity.

Complete genome characterization of Genogroup II norovirus strains from India: Evidence of recombination in ORF2/3 overlap

Noroviruses (NoVs) are considered as important causative agents of non-bacterial acute gastroenteritis, worldwide. The data on NoV genomes, their diversity and evolution from Indian subcontinent are not available to date. The present study describes the characterization of full-length genomes of Indian NoV strains for the first time to establish their phylogenetic and evolutionary relationship with those circulating worldwide. Amplification of full-length genomes of three NoV strains (PC15, PC51 and PC52) was carried out using nine overlapping sets of forward and reverse primers. Full-length genomes of all of the three strains were characterized by phylogenetic, SimPlot, selection pressure and hydrophilicity analyses. The strain, PC15 was placed in the GII.4-Hunter subcluster. An intragenotype recombination event between ORFs 2 (new GII.4 variant) and 3 (Den Haag subcluster) of the strain, PC51 was detected for the first time in this study. The strain, PC52 showed the presence of commonly detected intergenotype recombination, GII.b/GII.3. A 16 amino-acid signature code (TDVVYYAGASQPRDDI) was identified in the ORF2 of recombinant GII.3 specificity strains, which may serve as a genetic marker for differentiation of these strains from non-recombinant GII.3 strains. The amino-acid substitutions in the ORF2 of PC51 and PC52 strains in comparison to the reference strains (Toyama1 and TV24) resulted in an increase in the hydrophilicity suggested alterations in the antigenic regions of Indian NoV strains. A unique pattern of amino-acid substitutions was observed within seven subclusters of GII.4 at 19 sites (including 13 sites under positive selection pressure) spanning entire ORF2. The study indicates adaptation of NoVs in the environment to escape the host immune response and to persist in the population. It also provides in-depth analyses of NoV genomes from India and determines the extent of conserved and variable features of the Indian NoV strains.

Norovirus genotype IIb associated acute gastroenteritis in India

BACKGROUND In India the role of noroviruses in causing gastroenteritis is not well defined. OBJECTIVE To determine the norovirus prevalence in sporadic cases of acute gastroenteritis in western India. STUDY DESIGN A total of 236 fecal specimens consisting 192 specimens from hospitalized and 44 from OPD (outpatient department) children < or = 5 years of age were tested for the presence of norovirus genogroup (G)I and GII by RT-PCR followed by sequencing. Multiple alignment and phylogenetic analysis were carried out to determine the norovirus genetic types. RESULTS Norovirus GII infection was detected in 11.9% (28/236) specimens. These included 24 of 192 (12.5%) specimens from hospitalized and 4 of 44 (9.0%) specimens from OPD cases. Nucleotide sequence based analysis of norovirus GII strains indicated circulation of GIIb strains at a significant level in hospitalized (12.5%) and OPD (75%) children. CONCLUSION This study demonstrates the predominance of GII/4 (genogroup II/genotype 4) along with co-circulation of GII/1, GII/2, GII/3 and GIIb revealing the possibility of norovirus as the second most common cause of non-bacterial acute gastroenteritis after rotavirus in western India. It also documents for the first time occurrence of norovirus GIIb infections in India.

Genetic and Epidemiologic Trends of Norovirus Outbreaks in the United States from 2013 to 2016 Demonstrated Emergence of Novel GII.4 Recombinant Viruses

ABSTRACT Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

Diversity in the Enteric Viruses Detected in Outbreaks of Gastroenteritis from Mumbai, Western India

Faecal specimens collected from two outbreaks of acute gastroenteritis that occurred in southern Mumbai, India in March and October, 2006 were tested for seven different enteric viruses. Among the 218 specimens tested, 95 (43.6%) were positive, 73 (76.8%) for a single virus and 22 (23.2%) for multiple viruses. Single viral infections in both, March and October showed predominance of enterovirus (EV, 33.3% and 40%) and rotavirus A (RVA, 33.3% and 25%). The other viruses detected in these months were norovirus (NoV, 12.1% and 10%), rotavirus B (RVB, 12.1% and 10%), enteric adenovirus (AdV, 6.1% and 7.5%), Aichivirus (AiV, 3% and 7.5%) and human astrovirus (HAstV, 3% and 0%). Mixed viral infections were largely represented by two viruses (84.6% and 88.9%), a small proportion showed presence of three (7.7% and 11%) and four (7.7% and 0%) viruses in the two outbreaks. Genotyping of the viruses revealed predominance of RVA G2P[4], RVB G2 (Indian Bangladeshi lineage), NoV GII.4, AdV-40, HAstV-8 and AiV B types. VP1/2A junction region based genotyping showed presence of 11 different serotypes of EVs. Although no virus was detected in the tested water samples, examination of both water and sewage pipelines in gastroenteritis affected localities indicated leakages and possibility of contamination of drinking water with sewage water. Coexistence of multiple enteric viruses during the two outbreaks of gastroenteritis emphasizes the need to expand such investigations to other parts of India.

Genetic diversity of genotype G1 rotaviruses co-circulating in western India

The rotavirus (RV) G1 strains represent the common genotype that causes diarrhea in humans. The aim of this study was to determine the genetic lineages of G1 RV strains circulating in western India during two different time periods, 1991-1994 and 2006 by molecular characterization of VP7 genes. The phylogenetic analysis of VP7 genes showed clustering of G1 strains into lineages I (96.4%) and VIII (3.6%) in 1991-1994 and I (96.2%) and II (3.8%) in 2006. The sublineage IA was predominant (96.4%) in the years 1991-1994, however, was detected only in 44.4% of the strains in 2006 co-circulating with other sublineages IB (44.4%), IC (3.7%), IE (3.7%) and IIB (3.7%). The amino acid substitutions were noted in the previously identified signature codes of sublineages IB and IIB at positions 75 and 55, respectively. The differentiation marker (Q) described for sublineage IB at position 16 was replaced by I in all Indian strains clustered in sublineage IB. The study reports the characterization of G1 RV strains on the basis of distinct lineages and sublineages from India and emphasizes continuous monitoring on the diversity of G1 strains across the Indian population.